Search Results (49)

Sweet taste receptors (STRs) are class C G protein-coupled receptors (GPCRs) that function as heterodimers of TAS1R2 and TAS1R3. These receptors possess multiple binding sites and can be activated by a wide range of sweet-tasting compounds. Interestingly, TAS1R2 alone or even its extracellular domain-truncated form (TAS1R2-TMD), can act as a functional receptor. Previous studies demonstrated that the sweetener S819 and the sweet inhibitor amiloride act through the transmembrane domain (TMD) of TAS1R2; however, the molecular mechanisms underlying these ligand-specific effects remain unclear, largely due to the historical lack of experimentally determined full-length STR structures. Recent breakthroughs in cryo-EM structural determination of the full-length TAS1R2/TAS1R3 complex now offer an unprecedented opportunity to elucidate receptor activation mechanisms at atomic resolution. In this study, we investigated ligand-induced conformational dynamics of hTAS1R2-TMD using microsecond-scale molecular dynamics (MD) simulations on three systems: hTAS1R2-TMD/S819 (agonist-bound), hTAS1R2-TMD/amiloride (antagonist-bound), and hTAS1R2-TMD (apo). Comparative analyses revealed that agonist and antagonist binding distinctly modulate key structural switches, including the conserved ionic lock (E6.35-R3.50), which stabilizes the inactive state and disrupts upon activation. Notably, we identified a novel salt bridge (D7.32-R3.32) that forms preferentially in the active state, potentially serving as a unique molecular switch for TAS1R2. Additional analyses uncovered ligand-specific rearrangements in hydrogen-bonding and hydrophobic interaction networks. These results provide atomistic insights into how agonists and antagonists differentially modulate TAS1R2 activation and lay a structural foundation for designing novel sweeteners and taste modulators. Full article

The invertebrate neuropeptide F (NPF) signaling plays versatile roles in diverse biological activities and processes. Still, whether and how it mediates feeding and digestion in Pomacea canaliculate remain gaps in our knowledge. Herein, we first identified and characterized PcNPFR via bioinformatics analysis in P. canaliculate, which is a polyphagous herbivore with a voracious appetite that causes devastating damages to ecosystem functioning and services in colonized ranges. Double stranded RNA (dsRNA)-based RNA interference (RNAi) and exogenous rescue were utilized to decipher and substantiate underlying mechanisms whereby NPFR executed its modulatory functions. Multiple sequence alignment and phylogeny indicated that PcNPFR harbored typical seven transmembrane domains (7 TMD) and belonged to rhodopsin-like GPCRs, with amino acid sequence sharing 27.61–63.75% homology to orthologues. Spatio-temporal expression profiles revealed the lowest abundance of PcNPFR occurred in pleopod tissues and the egg stage, while it peaked in male snails and testes. Quantitative real-time PCR (qRT-PCR) analysis showed that 4 µg dsNPFR and 10⁻⁶ M trNPF (NPFR agonist) were optimal doses to exert silencing and rescue effects, accordingly with sampling time at 3 days post treatments. Moreover, the dsNPFR injection (4 µg) at 1/3/5/7 day/s delivered silencing efficiency of 32.20–74.01%. After 3 days upon dsNPFR knockdown (4 µg), mRNA levels of ILP7/InR/Akt/PI3Kc/PI3K_R were significantly downregulated compared to dsGFP controls, except FOXO substantially upregulated at both transcript and translation levels. In addition, the activities of alpha-amylase, protease and lipase were significantly suppressed, accompanied by decreased leaf area consumption, attenuated feeding behavior and diminished feeding rate. Moreover, expression trends were opposite and proxies were partially or fully restored to baseline levels post exogenous compensation of trNPF, suggesting phenotypes specifically attributable to PcNPFR RNAi but not off-target effects. PcNPFR is implicated in both feeding and digestion by modulating the ISP pathway and digestive enzyme activities. It may serve as a promising molecular target for RNAi-based antifeedants to manage P. canaliculate invasion. Full article

N-methyl-D-aspartate receptors (NMDARs) are critical components of the mammalian central nervous system, involved in synaptic transmission, plasticity, and neurodevelopment. This review focuses on the structural and functional characteristics of NMDARs, with a particular emphasis on the GRIN2 subunits (GluN2A-D). The diversity of GRIN2 subunits, driven by alternative splicing and genetic variants, significantly impacts receptor function, synaptic localization, and disease manifestation. The temporal and spatial expression of these subunits is essential for typical neural development, with each subunit supporting distinct phases of synaptic formation and plasticity. Disruptions in their developmental regulation are linked to neurodevelopmental disorders, underscoring the importance of understanding these dynamics in NDD pathophysiology. We explore the physiological properties and developmental regulation of these subunits, highlighting their roles in the pathophysiology of various NDDs, including ASD, epilepsy, and schizophrenia. By reviewing current knowledge and experimental models, including mouse models and human-induced pluripotent stem cells (hiPSCs), this article aims to elucidate different approaches through which the intricacies of NMDAR dysfunction in NDDs are currently being explored. The comprehensive understanding of NMDAR subunit composition and their mutations provides a foundation for developing targeted therapeutic strategies to address these complex disorders. Full article

Human Immunodeficiency Virus (HIV) is a diploid, C-type enveloped retrovirus belonging to the Lentivirus genus, characterized by two positive-sense single-stranded RNA genomes, that transitioned from non-human primates to humans and has become globally widespread. In its advanced stages, HIV leads to Acquired Immune Deficiency Syndrome (AIDS), which severely weakens the immune system by depleting CD4+ helper T cells. Without treatment, HIV progressively impairs immune function, making the body susceptible to various opportunistic infections and complications, including cardiovascular, respiratory, and neurological issues, as well as secondary cancers. The envelope glycoprotein complex (Env), composed of gp120 and gp41 subunits derived from the precursor gp160, plays a central role in cycle entry. gp160, synthesized in the rough endoplasmic reticulum, undergoes glycosylation and proteolytic cleavage, forming a trimeric spike on the virion surface. These structural features, including the transmembrane domain (TMD), membrane-proximal external region (MPER), and cytoplasmic tail (CT), are critical for viral infectivity and immune evasion. Glycosylation and proteolytic processing, especially by furin, are essential for Env’s fusogenic activity and capacity to evade immune detection. The virus’s outer envelope glycoprotein, gp120, interacts with host cell CD4 receptors. This interaction, along with the involvement of coreceptors CXCR4 and CCR5, prompts the exposure of the gp41 fusogenic components, enabling the fusion of viral and host cell membranes. While this is the predominant pathway for viral entry, alternative mechanisms involving receptors such as C-type lectin and mannose receptors have been found. This review aims to provide an in-depth analysis of the structural features and functional roles of HIV entry proteins, particularly gp120 and gp41, in the viral entry process. By examining these proteins’ architecture, the review elucidates how their structural properties facilitate HIV invasion of host cells. It also explores the synthesis, trafficking, and structural characteristics of Env/gp160 proteins, highlighting the interactions between gp120, gp41, and the viral matrix. These contributions advance drug resistance management and vaccine development efforts. Full article

Feline calicivirus (FCV), an important model for studying the biology of the Caliciviridae family, encodes the leader of the capsid (LC) protein, a viral factor known to induce apoptosis when expressed in a virus-free system. Our research has shown that the FCV LC protein forms disulfide bond-dependent homo-oligomers and exhibits intrinsic toxicity; however, it lacked a polybasic region and a transmembrane domain (TMD); thus, it was initially classified as a non-classical viroporin. The unique nature of the FCV LC protein, with no similarity to other proteins beyond the Vesivirus genus, has posed challenges for bioinformatic analysis reliant on sequence similarity. In this study, we continued characterizing the LC protein using the AlphaFold 2 and the recently released AlphaFold 3 artificial intelligence tools to predict the LC protein tertiary structure. We compared it to other molecular modeling algorithms, such as I-Tasser’s QUARK, offering new insights into its putative TMD. Through exogenous interaction, we found that the recombinant LC protein associates with the CrFK plasmatic membrane and can permeate cell membranes in a disulfide bond-independent manner, suggesting that this interaction might occur through a TMD. Additionally, we examined its potential to activate the intrinsic apoptosis pathway in murine and human ovarian cancer cell lines, overexpressing survivin, an anti-apoptotic protein. All these results enhance our understanding of the LC protein’s mechanism of action and suggest its role as a class-I viroporin. Full article

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium–high resolution of 2–4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling. Full article

Recently, several ATP-binding cassette (ABC) importers have been found to adopt the typical fold of type IV ABC exporters. Presumably, these importers would function under the transport scheme of “alternating access” like those exporters, cycling through inward-open, occluded, and outward-open conformations. Understanding how the exporter-like importers move substrates in the opposite direction requires structural studies on all the major conformations. To shed light on this, here we report the structure of yersiniabactin importer YbtPQ from uropathogenic Escherichia coli in the occluded conformation trapped by ADP-vanadate (ADP-Vi) at a 3.1 Å resolution determined by cryo-electron microscopy. The structure shows unusual local rearrangements in multiple helices and loops in its transmembrane domains (TMDs). In addition, the dimerization of the nucleotide-binding domains (NBDs) promoted by the vanadate trapping is highlighted by the “screwdriver” action at one of the two hinge points. These structural observations are rare and thus provide valuable information to understand the structural plasticity of the exporter-like ABC importers. Full article

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme transforming vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents delivered by its redox partner through thiol-disulfide exchange reactions. The luminal loop (L-loop) is the principal mediator of hVKORC1 activation, and it is a region frequently harbouring numerous missense mutations. Four L-loop hVKORC1 mutants, suggested in vitro as either resistant (A41S, H68Y) or completely inactive (S52W, W59R), were studied in the oxidised state by numerical approaches (in silico). The DYNASOME and POCKETOME of each mutant were characterised and compared to the native protein, recently described as a modular protein composed of the structurally stable transmembrane domain (TMD) and the intrinsically disordered L-loop, exhibiting quasi-independent dynamics. The DYNASOME of mutants revealed that L-loop missense point mutations impact not only its folding and dynamics, but also those of the TMD, highlighting a strong mutation-specific interdependence between these domains. Another consequence of the mutation-induced effects manifests in the global changes (geometric, topological, and probabilistic) of the newly detected cryptic pockets and the alternation of the recognition properties of the L-loop with its redox protein. Based on our results, we postulate that (i) intra-protein allosteric regulation and (ii) the inherent allosteric regulation and cryptic pockets of each mutant depend on its DYNASOME; and (iii) the recognition of the redox protein by hVKORC1 (INTERACTOME) depend on their DYNASOME. This multifaceted description of proteins produces “omics” data sets, crucial for understanding the physiological processes of proteins and the pathologies caused by alteration of the protein properties at various “omics” levels. Additionally, such characterisation opens novel perspectives for the development of “allo-network drugs” essential for the treatment of blood disorders. Full article

KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment. Full article

Herpesvirus entry requires the coordinated action of at least four viral glycoproteins. Virus-specific binding to a cellular receptor triggers a membrane fusion cascade involving the conserved gH/gL complex and gB. Although gB is the genuine herpesvirus fusogen, it requires gH/gL for fusion, but how activation occurs is still unclear. To study the underlying mechanism, we used a gL-deleted pseudorabies virus (PrV) mutant characterized by its limited capability to directly infect neighboring cells that was exploited for several independent serial passages in cell culture. Unlike previous revertants that acquired mutations in the gL-binding N-terminus of gH, we obtained a variant, PrV-ΔgLPassV99, that unexpectedly contained two amino acid substitutions in the gH transmembrane domain (TMD). One of these mutations, I662S, was sufficient to compensate for gL function in virus entry and in in vitro cell–cell fusion assays in presence of wild type gB, but barely for cell-to-cell spread. Additional expression of receptor-binding PrV gD, which is dispensable for cell–cell fusion mediated by native gB, gH and gL, resulted in hyperfusion in combination with gH V99. Overall, our results uncover a yet-underestimated role of the gH TMD in fusion regulation, further shedding light on the complexity of herpesvirus fusion involving all structural domains of the conserved entry glycoproteins. Full article

Membrane-spanning portions of proteins’ polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an “anchor” in ensuring the correct orientation of the protein’s extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of “the protein machine”. They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike’s fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins’ TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses. Full article

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability. Full article

The copper transporter (COPT/Ctr) gene family plays a critical part in maintaining the balance of the metal, and many diverse species depend on COPT to move copper (Cu) across the cell membrane. In Arabidopsis thaliana, Oryza sativa, Medicago sativa, Zea mays, Populus trichocarpa, Vitis vinifera, and Solanum lycopersicum, a genome-wide study of the COPT protein family was performed. To understand the major roles of the COPT gene family in Kandelia obovata (Ko), a genome-wide study identified four COPT genes in the Kandelia obovata genome for the first time. The domain and 3D structural variation, phylogenetic tree, chromosomal distributions, gene structure, motif analysis, subcellular localization, cis-regulatory elements, synteny and duplication analysis, and expression profiles in leaves and Cu were all investigated in this research. Structural and sequence investigations show that most KoCOPTs have three transmembrane domains (TMDs). According to phylogenetic research, these KoCOPTs might be divided into two subgroups, just like Populus trichocarpa. KoCOPT gene segmental duplications and positive selection pressure were discovered by universal analysis. According to gene structure and motif analysis, most KoCOPT genes showed consistent exon–intron and motif organization within the same group. In addition, we found five hormones and four stress- and seven light-responsive cis-elements in the KoCOPTs promoters. The expression studies revealed that all four genes changed their expression levels in response to copper (CuCl₂) treatments. In summary, our study offers a thorough overview of the Kandelia obovata COPT gene family’s expression pattern and functional diversity, making it easier to characterize each KoCOPT gene’s function in the future. Full article

Intramembrane proteases, such as γ secretase, typically recruit multiple substrates from an excess of single-span membrane proteins. It is currently unclear to which extent substrate recognition depends on specific interactions of their transmembrane domains (TMDs) with TMDs of a protease. Here, we investigated a large number of potential pairwise interactions between TMDs of γ secretase and a diverse set of its substrates using two different configurations of BLaTM, a genetic reporter system. Our results reveal significant interactions between TMD2 of presenilin, the enzymatic subunit of γ secretase, and the TMD of the amyloid precursor protein, as well as of several other substrates. Presenilin TMD2 is a prime candidate for substrate recruitment, as has been shown from previous studies. In addition, the amyloid precursor protein TMD enters interactions with presenilin TMD 4 as well as with the TMD of nicastrin. Interestingly, the Gly-rich interfaces between the amyloid precursor protein TMD and presenilin TMDs 2 and 4 are highly similar to its homodimerization interface. In terms of methodology, the economics of the newly developed library-based method could prove to be a useful feature in related future work for identifying heterotypic TMD−TMD interactions within other biological contexts. Full article

Multidrug resistance (MDR) proteins belonging to the ATP-Binding Cassette (ABC) transporter group play a crucial role in the export of cytotoxic drugs across cell membranes. These proteins are particularly fascinating due to their ability to confer drug resistance, which subsequently leads to the failure of therapeutic interventions and hinders successful treatments. One key mechanism by which multidrug resistance (MDR) proteins carry out their transport function is through alternating access. This mechanism involves intricate conformational changes that enable the binding and transport of substrates across cellular membranes. In this extensive review, we provide an overview of ABC transporters, including their classifications and structural similarities. We focus specifically on well-known mammalian multidrug resistance proteins such as MRP1 and Pgp (MDR1), as well as bacterial counterparts such as Sav1866 and lipid flippase MsbA. By exploring the structural and functional features of these MDR proteins, we shed light on the roles of their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) in the transport process. Notably, while the structures of NBDs in prokaryotic ABC proteins, such as Sav1866, MsbA, and mammalian Pgp, are identical, MRP1 exhibits distinct characteristics in its NBDs. Our review also emphasizes the importance of two ATP molecules for the formation of an interface between the two binding sites of NBD domains across all these transporters. ATP hydrolysis occurs following substrate transport and is vital for recycling the transporters in subsequent cycles of substrate transportation. Specifically, among the studied transporters, only NBD2 in MRP1 possesses the ability to hydrolyze ATP, while both NBDs of Pgp, Sav1866, and MsbA are capable of carrying out this reaction. Furthermore, we highlight recent advancements in the study of MDR proteins and the alternating access mechanism. We discuss the experimental and computational approaches utilized to investigate the structure and dynamics of MDR proteins, providing valuable insights into their conformational changes and substrate transport. This review not only contributes to an enhanced understanding of multidrug resistance proteins but also holds immense potential for guiding future research and facilitating the development of effective strategies to overcome multidrug resistance, thus improving therapeutic interventions. Full article